Evidence for galanin as an inhibitory neuropeptide on myenteric cholinergic neurons in the guinea pig small intestine

The effect of galanin on the [3H]ACh release from myenteric plexus-longitudinal muscle strips of the guinea pig small intestine was studied. While galanin did not alter the basal spontaneous efflux of ACh, it significantly depressed the ACh release evoked by electrical stimulation or caused by VIP and substance P. These results suggest an important neuromodulatory role for galanin in the enteric nervous system.     

5-HT1B receptor regulation of serotonin (5-HT) release by endogenous 5-HT in the substantia nigra

Axonal release of serotonin (5-hydroxytryptamine, 5-HT) in the CNS is typically regulated by presynaptic 5-HT autoreceptors. Release of 5-HT in substantia nigra pars reticulata (SNr), a principal output from the basal ganglia, has seemed an interesting exception to this rule. The SNr receives one of the highest densities of 5-HT innervation in mammalian brain and yet negative feedback regulation of axonal 5-HT release by endogenous 5-HT has not been identified here. We explored whether we could identify autoregulation of 5-HT release by 5-HT_1B receptors in rat SNr slices using fast-scan cyclic voltammetry at carbon-fiber microelectrodes to detect 5-HT release evoked by discrete stimuli (50 Hz, 20 pulses) paired over short intervals (1–10 s) within which any autoreceptor control should occur. Evoked 5-HT release exhibited short-term depression after an initial stimulus that recovered by 10 s. Antagonists for 5-HT_1B receptors, isamoltane (1 μM) or SB 224-289 (1 μM), did not modify release during a stimulus train, but rather, they modestly relieved depression of subsequent release evoked after a short delay (≤2 s). Release was not modified by antagonists for GABA (picrotoxin, 100 μM, saclofen, 50 μM) or histamine-H₃ (thioperamide, 10 μM) receptors. These data indicate that 5-HT release can activate a 5-HT_1B-receptor autoinhibition of subsequent release, which is mediated directly via 5-HT axons and not via GABAergic or histaminergic inputs. These data reveal that 5-HT release in SNr is not devoid of autoreceptor regulation by endogenous 5-HT, but rather is under modest control which only weakly limits 5-HT signaling.     

Release of transmitters into the perfused third cerebral ventricle of the cat

1. The third cerebral ventricle of cats treated with nialamide and anaesthetized with chloralose was perfused, and the effluent was tested for 5-hydroxytryptamine (5-HT) and also for acetylcholine (ACh) when the perfusion fluid contained neostigmine.

2. Under `resting' conditions a 25 min sample of effluent contained from < 1 to 6 ng 5-HT; the release remained steady during many hours of perfusion. It was necessary to watch out for traces of blood which might contribute to the 5-HT content and which were only visible after centrifugation.

3. A number of regions in the ventral mid-brain and hind-brain were stimulated, including the two most anterior nuclei of the raphe, nucleus linearis rostralis and intermedius. Release of 5-HT (rarely more than 2 ng) was only obtained on stimulation of these two nuclei, whereas ACh was released by stimulating many points, such as the reticular formation or the decussation of the superior cerebellar peduncles, but not the two raphe nuclei.

4. Low frequencies of stimulation were more effective at releasing 5-HT, and high frequencies at releasing ACh.

5. Since the amount of 5-HT released on stimulation was rarely more than 2 ng, a powerful re-uptake process was suspected and confirmed by the use of chlorimipramine. Intravenous, intraperitoneal and intraventricular use of this drug temporarily increased the basal release to values ranging from 20 to 50 ng in 25 min samples, and about trebled the release on stimulation of either of the linear nuclei.

6. Intravenous administration of chlorimipramine (10 mg/kg) caused the disappearance of electrical responses evoked in the brain stem by afferent sensory stimuli.

     

MK-801 blocks nicotinic depolarizations of guinea pig myenteric neurons

Intracellular recordings were made from guinea-pig myenteric neurons. Acetylcholine (ACh), applied by iontophoresis, produced a depolarization that was blocked by hexamethonium (EC50 3.2 microM) but not by scopolamine (1 microM). MK-801 (0.3-30 microM), a non-competitive antagonist at N-methyl-D-aspartate receptors, also blocked the nicotinic depolarization (EC50 = 4.5 microM). Voltage clamp measurements of membrane current showed that MK-801 (1 microM) and hexamethonium (1 microM) both produced a greater inhibition of ACh-induced inward currents at -100 mV than at -40 mV. MK-801 (10 microM) did not change the depolarization evoked by 5-HT (acting at 5-HT3 receptors) in the same neurons in which it reduced the nicotinic response by more than 70%. It is concluded that MK-801 can act as a non-competitive antagonist of nicotinic responses in myenteric neurons.     

Pharmacology of pH effects on carotid body chemoreceptors in vitro

1. The carotid body and the carotid nerve were removed from anaesthetized cats and placed in a small Perspex channel through which Locke solution (at various pH values and usually equilibrated with 50% O(2) in N(2)) was allowed to flow. The glomus was immersed in the flowing solution while the nerve was lifted into oil covering the saline. Sensory discharges were recorded from the nerve and their frequency was used as an index of receptor activity. At times, a small segment of carotid artery, containing pressoreceptor endings, was removed together with the glomus. In this case, pressoreceptor discharges were recorded from the nerve.2. The amplitude of either chemo- or pressoreceptor discharges was not changed by strong acid solutions. Acid decreased the frequency of the baroreceptor discharges only when pH fell to less than 4.0. Solutions at low pH increased the chemosensory discharge, but acid depressed the increased chemoreceptor discharge elicited by KCl. These experiments indicated that H(+) ions probably acted as membrane ;stabilizers' without depolarizing either the nerve fibres or endings.3. Acid solutions increased the action of acetylcholine chloride (AChCl) (100-200 mug) on chemoreceptors. This effect probably was due either to inactivation of tissue cholinesterase or to enhanced sensitivity of the sensory endings to ACh.4. Choline chloride (10(-3)M), which favours ACh synthesis, protected the preparation against decay during prolonged experimentation. Hemicholinium-3 (HC-3), which blocks ACh synthesis in low concentrations (10(-5)M), depressed the chemosensory response to acid and to hypoxia when such stimuli were applied repeatedly. This concentration of HC-3 did not change effects of applied ACh.5. Substances which affect ACh release markedly changed the chemoreceptor discharge increase induced by acidity and other forms of stimulation. In the absence of Ca(2+), acid, anoxia, and interruption of flow provoked receptor depression while receptor excitation induced by ACh and KCl persisted. All stimuli excited and showed increased effectiveness as the Ca(2+) concentration was raised, but their effects declined as Ca(2+) was increased above normal values. Mg(2+) ions depressed the chemoreceptor effects induced by all these stimuli. The action of Mg(2+) was not due entirely to nerve ending block. Morphine sulphate (which decreases ACh release in other structures) also depressed the receptor response to acid and flow interruption.6. Cholinergic blocking agents such as mecamylamine, hexamethonium, atropine, dihydro-beta-erithroidine (DHE), HC-3 (10(-4)M), choline and acetylcholine (in combination with choline) depressed the effects of acid and ACh on the chemoreceptors. The effect induced by interruption of flow was depressed only by mecamylamine and DHE.7. Agents which affect the fate of released ACh, such as acetylcholinesterase and eserine salicylate, did not affect clearly the response of chemoreceptors to acid.8. The results suggest that acid stimulates chemoreceptor fibres through an indirect mechanism, viz. through increased release and/or decreased destruction of a presynaptic transmitter from the glomus cell. This transmitter is probably ACh (see following paper, Eyzaguirre & Zapata, 1968).     

Neuronal bursting induced by NK3 receptor activation in the neonatal rat spinal cord in vitro.

Intracellular recording from lumbar motoneurons and extracellular recording from ventral roots of the neonatal rat isolated spinal cord were used to study the mechanisms responsible for the excitation mediated by NK3 tachykinin receptors. The selective NK3 agonists senktide or [MePhe7]neurokinin B induced a slow depolarization with superimposed oscillations (mean period +/- SD was 2.8 +/- 0.8 s) that, in the majority of cases, showed left-right alternation at segmental level and were synchronous between L2 and L5 of the same side. During agonist wash out (5-20 min) a delayed form of hyperexcitability emerged consisting of bursts lasting 8 +/- 2 s (average interburst interval 55 +/- 21 s) with superimposed oscillations usually with homosegmental alternation and heterosegmental synchronicity. Such bursting was accompanied by depression of GABAergic dorsal root potentials evoked by dorsal root stimulation and of the recurrent inhibitory postsynaptic potential recorded from motoneurons. Despite bursting, motoneuron membrane potential returned to baseline while input resistance was increased. Bursts were a network-dependent phenomenon triggered by previous NK3 receptor activation because bursting was suppressed by glutamate receptor antagonists and was insensitive to motoneuron membrane potential or subsequent application of an NK3 receptor antagonist. NK3 receptors operated synergistically with N-methyl-D-aspartate (NMDA) and 5-hydroxytryptamine (5-HT) to trigger fully alternating locomotor-like rhythms while NK3 receptor antagonism disrupted the same rhythm. In summary, in the neonatal rat spinal cord NK3 receptors could trigger rhythmic activity predominantly with alternation at segmental level but with synchronous coupling between ipsilateral motor pools. NK3 receptor activation could also facilitate fictive locomotor patterns induced by NMDA and 5-HT.     

Substance P-evoked release of acetylcholine from isolated spinal cord of the newborn rat.

Isolated spinal cords of newborn rats were perfused with artificial cerebrospinal fluid and the release of endogenous acetylcholine was measured using high-performance liquid chromatography with an electrochemical detection system. Application of high-K+ (90 mM) medium evoked about an eight-fold increase in the acetylcholine release, and the K(+)-evoked release was Ca2+ dependent. Veratridine (20 microM) also evoked about a four-fold increase in the acetylcholine release, and this increase was suppressed by 0.2 microM tetrodotoxin. Application of substance P at 0.3-3 microM evoked a concentration-dependent release of acetylcholine. The substance P-evoked acetylcholine release was Ca2+ dependent and abolished by tetrodotoxin. Neurokinin A, neurokinin B, acetyl-Arg6-septide and senktide (3 microM each) also evoked a release of acetylcholine. Electrophysiological experiments using isolated spinal cords of newborn rats showed that bath application of substance P induced a depolarization of motoneurons, which was enhanced by edrophonium. This enhancement of substance P-induced depolarization by edrophonium disappeared in a low-Ca2+ medium or in the presence of atropine and dihydro-beta-erythroidine. In the presence of edrophonium and dihydro-beta-erythroidine, substance P induced an inhibition of monosynaptic reflex, and this inhibition was abolished by atropine. These results suggest that substance P and other tachykinins induce a release of acetylcholine from the newborn rat spinal cord by exciting cholinergic neurons.     

Endogenous glutamatergic synaptic activity elicits acetylcholine release from rat cultured septal cells

We tested the characteristics of acetylcholine (ACh) release from cultured rat septal cells. The spontaneous release was inhibited by treatment with tetrodotoxin (TTX) and omega-conotoxin (GVIA), indicating that the release was elicited by synaptic activity. The release was also inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor blocker, in both the absence and presence of nerve growth factor (NGF), suggesting that endogenously released glutamate produced the ACh release by stimulating AMPA receptors.This is the first report of detection of the release of ACh by endogenous spontaneous synaptic activity conducted by glutamate AMPA receptor activation in cultured septal cells. This in vitro experimental system is useful for the study of cholinergic functions.     

Senktide, a selective neurokinin B-like agonist, elicits serotonin-mediated behaviour following intracisternal administration in the mouse

Behavioural responses to tachykinins were observed following intracisternal administration in mice. The synthetic NK-3 agonists senktide and L-363,851 caused behaviour typically associated with serotonergic stimulation, including head twitches, reciprocal forepaw treading and hindlimb splaying. Neurokinin B produced some features of the serotonin (5-HT) syndrome, while substance P, neurokinin A and eledoisin failed to elicit any such behaviours. Senktide-induced head twitches were prevented by pretreatment with the 5-HT₂ antagonists ketanserin and ritanserin, while forepaw treading was attenuated by the 5-HT₁ antagonists (−)-pindolol and methysergide. These data suggest that NK-3 agonists interact with central 5-HT mechanisms.     

Effect of hydroxycitric acid on serotonin release from isolated rat brain cortex.

There is evidence that hydroxycitric acid (HCA), an extract of dried fruit rind of South Asian trees of the genus Garcinia cambogia, can reduce food intake in experimental animals. In the present study, we investigated the effect of HCA on basal and potassium-depolarization evoked increase in radiolabeled serotonin ([3H]-5-HT) release from rat brain cortex slices in vitro. HCA (10 microM-1 mM) altered the baseline of spontaneous tritium efflux but had no significant effect on potassium-evoked release of [3H]-5-HT. When applied on its own, HCA (10 microM-1 mM) elicited a concentration-dependent increase in efflux of [3H]-5-HT reaching a maximum at 300 microM. We conclude that HCA can increase the release of radiolabeled 5-HT from the isolated rat brain cortex.     

Long term effects of septohippocampal lesions and intrahippocampal grafts on acetylcholine concentration,muscarinic stimulated formation of inositol phospholipids and electrically evoked release of neurotransmitters in the rat hippocampus

Summary Long Evans female rats sustained aspirative lesions of the septohippocampal pathways; subsequently, they received intrahippocampal suspension grafts of fetal septal-diagonal band or hippocampal tissue. The long term (8–10 months post-surgery) effects of these treatments were examined in the hippocampus for the following variables: concentration of hippocampal acetylcholine (ACh), muscarinic-stimulated (carbachol) formation of inositol monophosphate, accumulation of tritiated choline, noradrenaline (³H-NA) and serotonin (³H-5-HT), electrically evoked release of ³H-acetylcholine (³H-ACh), ³H-NA and ³H-5-HT, and choline acetyltransferase (ChAT) activity. The lesions decreased the levels of endogeneous ACh, the accumulation of ³H-choline and ³H-5-HT and the evoked release of both ³H-ACh and ³H-5-HT as well as the ChAT activity, but they failed to significantly affect the muscarinic-stimulated formation of inositol monophosphate and the accumulation and release of ³H-NA. Grafts of hippocampal cells were found to be ineffective on all lesion-induced effects. In contrast, grafts of septal-diagonal band origin attenuated the deficit of hippocampal concentrations of ACh and accumulation of ³H-choline without, however, improving release of ³H-ACh, accumulation and release of ³H-5-HT, and ChAT activity. These observations suggest that: (i) denervation-induced hippocampal muscarinic supersensitivity might not be long-lasting or the lesions, which in some cases spared the lateral edges of the fimbria, failed to induce any muscarinic supersensitivity, (ii) intrahippocampal grafts rich in cholinergic neurons do not foster recovery from the lesion-induced noncholinergic deficits we assessed, (iii) recovery of function may be expressed by some but not all biochemical or pharmacological cholinergic variables and (iv) graft-derived hippocampal reinnervation may be less efficient than the endogenous innervation of intact rats as indicated by the restoration of only some of the variables related to cholinergic function by intrahippocampal septal-diagonal band grafts.     

Release of catecholamines and dopamine beta-hydroxylase from the perfused adrenal gland of the cat.

1. Secretion of catecholamines (CA) and dopamine beta-hydroxylase (DBH) activity from the perfused cat adrenal gland was studied following splanchnic nerve stimulation or infusion of acetylcholine (ACh). 2. Splanchnic nerve stimulation (30 Hz) or perfusion with a low concentration of ACh (10-minus5 M) caused a marked release of CA in the venous effluent, but release of DBH activity was minimal while a higher concentration of ACh (10-minus 4 M) enhanced the release of CA and DBH. 3. The ratio of DBH/CA released in the perfusate by splanchnic nerve stimulation or ACh infusion was only a small fraction of the ratio in the soluble lysate of purified chromaffin vesicles. 4. Following reserpine treatment, adrenal CA levels fell to 25% of the control value in 24 hr, remained depressed on days 2, 3, 4 and 5 at 5% of the control and recovered to 60% of the control value on the 6th day. DBH activity was unchanged from the control value at 24 hr after treatment, then rose as high as 5 times the control on the 5th day and was still twice the control value on the 6th day. 5. CA secretion in response to ACh (10-minus 4 M) perfusion was reduced to 30% of the control value on the first day after reserpine treatment, while DBH secretion was unchanged. On the 2nd day, CA secretion was depressed further to 5% of the control and remained at this low level up to 5 days after treatment while DBH secretion was twice the control value at 48 hr and then on days 3, 4 and 5 rose up to 5 times the control value. On the 6th day, secretion of CA recovered to 30% of the control while DBH secretion was now twice the control. 6. Isopycnic sucrose density (discontinuous) gradient centrifugation of vesicles from adrenal glands of control cats, and of cats given reserpine 1 or 2 days perviously, indicated that new vesicles or vesicles depleted of CA by reserpine had a lower equilibrium density than the original population of vesicles. 7. These results suggest that the release of CA is quantal in nature, but the release of DBH is not necessarily coupled with it. Release of DBH by ACh from reserpinized glands suggests that the vesicles which were once involved in secretion may be re-used for synthesis and storage of CA.     

GABA(A) receptor facilitation of neurokinin release from primary afferent terminals in the rat spinal cord

Our goal was to test the following hypotheses: 1) GABA_A receptors facilitate neurokinin release from primary afferent terminals; 2) they do this by suppressing an inhibitory effect of GABA_B receptors; 3) the activation of these two receptors is controlled by the firing frequency of primary afferents. We evoked neurokinin release by stimulating the dorsal root attached to spinal cord slices, and measured it using neurokinin 1 receptor (NK1R) internalization. Internalization evoked by root stimulation at 1 Hz (but not at 100 Hz) was increased by the GABA_A receptor agonists muscimol (effective concentration of drug for 50% of the increase [EC₅₀] 3 μM) and isoguvacine (EC₅₀ 4.5 μM). Internalization evoked by root stimulation at 100 Hz was inhibited by the GABA_A receptor antagonists bicuculline (effective concentration of drug for 50% of the inhibition [IC₅₀] 2 μM) and picrotoxin (IC₅₀ 243 nM). Internalization evoked by incubating the root with capsaicin (to selectively recruit nociceptive fibers) was increased by isoguvacine and abolished by picrotoxin. Therefore, GABA_A receptors facilitate neurokinin release. Isoguvacine-facilitated neurokinin release was inhibited by picrotoxin, low Cl⁻, low Ca²⁺, Ca²⁺ channel blockers and N-methyl-d-aspartate receptor antagonists. Bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter, inhibited isoguvacine-facilitated neurokinin release, but this could be attributed to a direct inhibition of GABA_A receptors. The GABA_B agonist baclofen inhibited NK1R internalization evoked by 100 Hz root stimulation (IC₅₀ 1.5 μM), whereas the GABA_B receptor antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP-55845) increased NK1R internalization evoked by 1 Hz root stimulation (EC₅₀ 21 nM). Importantly, baclofen inhibited isoguvacine-facilitated neurokinin release, and CGP-55845 reversed the inhibition of neurokinin release by bicuculline. In conclusion, 1) GABA_B receptors located presynaptically in primary afferent terminals inhibit neurokinin release; 2) GABA_A receptors located in GABAergic interneurons facilitate neurokinin release by suppressing GABA release onto these GABA_B receptors; 3) high frequency firing of C-fibers stimulates neurokinin release by activating GABA_A receptors and inhibiting GABA_B receptors, whereas low frequency firing inhibits neurokinin release by the converse mechanisms.     

GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways

GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca2+ concentration of guinea-pig myenteric neurons (at 35 degrees C) using Fura-2-AM. Neurons were identified by 75 mM K+ depolarization (5 s), which evoked a transient intracellular Ca2+ concentration increase. GABA (10 s) induced a dose dependent (5 nM-1 microM) transient intracellular Ca2+ concentration rise in the majority of neurons (500 nM GABA: 251+/-17 nM, n=232/289). Interestingly, the response to 5 microM GABA (n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly (P<0.001) reduced by GABAA and GABAB receptor antagonists (10 microM) bicuculline and phaclofen. The GABAA agonist isoguvacine (10 microM) and GABAB agonist baclofen (10 microM) induced similar responses as 50 nM GABA, while the GABAC agonist cis-4-aminocrotonic acid (CACA) (10 microM) only elicited small responses in a minority of neurons. Removal of extracellular Ca2+ abolished all responses while depletion of intracellular Ca2+ stores by thapsigargin (5 microM) did not alter the responses to 500 nM GABA (n=13), but reduction of Ca2+ influx through voltage-dependent Ca2+ channels did. The nicotinic antagonist hexamethonium (100 microM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and the 5-HT3 receptor blocker ondansetron only had minor effects. Conclusion: GABA elicits transient intracellular Ca2+ concentration responses in the majority of myenteric neurons through activation of GABAA and GABAB receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5 microM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.     

5-Hydroxytryptamine 1B receptors mediate presynaptic inhibition of monosynaptic IPSC in the rat dorsolateral septal nucleus

Effects of 5-hydroxytryptamine (5-HT) on inhibitory synaptic transmission in the rat dorsolateral septal nucleus (DLSN) were examined by conventional intracellular and voltage-clamp recording methods. 5-HT (1-30 microM) depressed the monosynaptic fast IPSC evoked by local stimulation of the DLSN in the presence of DNQX, AP5 and CGP 55845A. CP 93129, a selective 5-HT1B receptor agonist, depressed the fast IPSC. The 5-HT-induced depression of the fast IPSC was attenuated by SB 216641, a selective antagonist for 5-HT1B receptors. 5-HT did not change the inward currents mediated by GABAA receptors, suggesting that 5-HT presynaptically inhibited the fast IPSC. 5-HT and CP 93129 depressed the frequency of miniature fast IPSPs (mIPSPs) without changing their amplitudes. Neither a selective protein kinase A inhibitor, H-89, nor a selective protein kinase C inhibitor, calphostin C, blocked the 5-HT-induced depression of the fast IPSC. N-Ethylmaleimide (NEM) blocked the 5-HT-induced depression of the evoked IPSC. These results suggest that activation of presynaptic 5-HT1B receptors depresses the release of GABA via a pertussis toxin (PTX)-sensitive G-protein in the rat DLSN.     

Peptidergic inhibition of cholinergic transmission in bullfrog sympathetic ganglia

Intracellular and voltage-clamp recordings were made from sympathetic B neurons to investigate an interaction between peptidergic and cholinergic responses in bullfrog sympathetic ganglia. Stimulations of both 3rd-5th (0.2 Hz) and 8th (30 Hz) spinal nerves evoked the fast excitatory postsynaptic potential (EPSP) superimposed with the late slow EPSP at the same sympathetic neuron. The amplitude of fast EPSPs was decreased during the course of the late slow EPSP in a majority of sympathetic neurons. The mean depression of the fast EPSP amplitude was 51 +/- 4% (n = 24). The quantal content of the fast EPSP was also depressed by 54 +/- 3% (n = 10) during the late slow EPSP. Acetylcholine-induced depolarization (ACh potential) and current (ACh current) produced by an ionophoretic application of ACh were not reduced during the late slow EPSP. Bath-application of LH-RH (40 nM-4 microM) depressed the fast EPSP in a concentration-dependent manner; at a concentration of 1 microM, it produced a 63 +/- 8% (n = 8) depression of the quantal content of the fast EPSP. LH-RH (1-4 microM) depressed the frequency of the miniature (M) EPSPs by 25 +/- 4% (n = 5) of control. Antagonists for luteinizing hormone-releasing hormone (LH-RH) receptor, [D-Phe2,6, Pro3]-LH-RH and [D-pGlu1, D-Phe2, D-Trp3,6]-LH-RH, prevented the presynaptic inhibition of the fast EPSP induced by LH-RH. These results suggest that the fast EPSP is depressed during the late slow EPSP by decreasing the evoked release of ACh from presynaptic nerve terminals in bullfrog sympathetic ganglia.     

Failure of omega-conotoxin to block L-channels associated with [3H]5-HT release in rat brain slices.

The effects of the mixed N- and L-type voltage-sensitive calcium channel (VSCC) antagonist, omega-conotoxin GVIA and the L-type VSCC agonist Bay K-8644 on calcium-dependent, potassium evoked release of [3H]5-hydroxtryptamine ([3H]5-HT) were investigated in slices of rat hippocampus. Bay K-8644 (1 microM) enhanced, whilst omega-conotoxin (10-30 nM) attenuated, but did not abolish, evoked release of [3H]5-HT. The facilitatory actions of Bay K-8644 on evoked release were unaffected by concentrations of omega conotoxin that significantly inhibited [3H]5-HT release. The experiments indicate that concentrations of omega-conotoxin which inhibit neurotransmitter release by blockade of N-type VSCC, may leave L-type calcium channel activity unaffected.     

Compensatory contribution of Cav2.3 channels to acetylcholine release at the neuromuscular junction of tottering mice

Tottering (Tg) mice carry the mutation P601L in their Cacna1a encoded Cav2.1 channels. Transmitter release at the wild-type neuromuscular junction (NMJ) is almost exclusively mediated by Cav2.1 channels, and we used this model synapse to study synaptic consequences of the Tg mutation. With electrophysiology, and using subtype-specific Cav2 channel-blocking toxins, we assessed a possible compensatory contribution of non-Cav2.1 channels to evoked acetylcholine (ACh) release at Tg NMJs. Release was reduced by approximately 75% by the Cav2.1 channel blocker omega-agatoxin-IVA, which was less than the approximately 95% reduction observed in wild-type. Release at Tg NMJs, but not at wild-type synapses, was reduced by approximately 15% by SNX-482, a Cav2.3 channel blocker. No Cav2.2 channel involvement was found. Probably, there is a small reduction in functional presynaptic Cav2.1 channels at Tg NMJs, which is compensated for by Cav2.3 channels. The remaining Cav2.1 channels are likely to convey enlarged Ca2+ flux, because evoked ACh release at Tg NMJs, at low extracellular Ca2+ concentration, was approximately sixfold higher than at wild-type NMJs. This is the first report of compensatory expression of non-Cav2.1 channels at NMJs of mice with a single amino acid change in Cav2.1.     

Adrenergic inhibition of serotonin release from neurons in guinea pig Auerback's plexus.

1. Norepinephrine reversibly blocked both the fast and slow stimulus-evoked EPSPs in myenteric ganglion cells. This action of norepinephrine was offset by the alpha-adrenergic blocking agent, phentolamine. 2. The putative neurotransmitter for the fast EPSP is acetylcholine, and serotonin (5-HT) is the suspected transmitter for the slow EPSP. 3. Norepinephrine did not block the excitatory effect of microiontophoresis of 5-HT onto the ganglion cells. 4. Norepinephrine decreased the input resistance and suppressed neuronal excitability. This effect was reversed by addition of 5-HT to the perfusion solution and was attributed to presynaptic blockade by norepinephrine of ongoing release of 5-HT. 5. Addition of norepinephrine after the slow EPSP had been evoked did not shorten the duration of the slow EPSP. 6. The results suggested that the mechanism of the blocking action of norepinephrine was inhibition of release of the excitatory neurotransmitters form presynaptic nerve terminals. This is probably one of the mechanisms that is involved in suppression of gastrointestinal motor function during activation of the sympathetic innervation of the bowel.